Previous studies have shown that supplementation of vitamin D3 to cow diets for 4 to 10 d before slaughter lowers Warner-Bratzler shear force (WBSF) values and increases sensory tenderness scores in beef cuts. The present study was conducted to evaluate the effects of vitamin D3 supplementation on muscle calcium concentration, WBSF values, and sensory tenderness ratings of LM and semitendinosus (ST) muscles from cull, predominately Angus, cows (eight cows per treatment). Treatments included 0 (control), 5 million IU, or 7.5 million IU of vitamin D3 supplemented daily for 7 d preslaughter. Twenty-four hours after slaughter, 2.54-cm-thick LM and ST muscle steaks were cut; aged for either 0, 7, 14, or 21 d (ST steaks aged for 7 d only); and frozen at -20 degrees C for WBFS and sensory analysis. Mean values for LM calcium concentration tended to increase (P = 0.14) with vitamin D3 supplementation (154, 176, and 183 microig/g, fresh basis, for 0, 5, and 7.5 million IU/d, respectively). After 7 d of aging, LM steaks from cows fed 7.5 million IU had lower (P < 0.05) WBSF values than 7-d steaks from controls and cows fed 5.0 million IU/d aged 7 d; however, vitamin D3 supplementation had no (P > 0.05) effect on WBSF values of ST steaks aged 7 d. Vitamin D3 supplementation did not (P > 0.05) affect sensory tenderness ratings for either LM or ST steaks at any aging period. Aging, however, had a linear (P < 0.001) effect on tenderness, with an increase in tenderness as aging time increased from 0 to 21 d. Thus, results from the present study indicate that vitamin D3 supplementation, at these levels and duration before slaughter, provided little benefit to muscle tenderness of beef from cull cows.